Protective Effects of Oil Palm Composition on Alzheimer&#39;s Disease

ABSTRACT

Present invention discloses an oil palm composition for use in prevention or treatment of Alzheimer&#39;s disease. The composition is useful in impeding formation of neurotoxic peptide. Present invention can be used in preparation of a medicament in a therapeutic effective amount for prevention or treatment of Alzheimer&#39;s disease and diseases related thereto.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage Application of InternationalApplication No. PCT/MY2014/000252, filed 10 Oct. 2014, which claimsbenefit of Serial No. PI 2013003739, filed 11 Oct. 2013 in Malaysia andwhich applications are incorporated herein by reference. To the extentappropriate, a claim of priority is made to each of the above disclosedapplications.

FIELD OF THE INVENTION

The present invention relates to a composition with protective effectstreating Alzheimer's disease. More particularly, relates to the use ofpalm oil-based phenolics in impeding β-amyloid peptide aggregation.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is an incurable neurodegenerative disease thatprogressively worsened and eventually led to death. AD is oftendiagnosed in persons over the age of 65 years and has affected 36million persons globally in 2010. At the current rate of increase inprevalence, it is predicted that AD will affect 1 in 85 personsworldwide by the year 2050.

The onset of AD often shows symptoms that appear to be “age-related”issues and difficulties in coping with stress. During the early stage,there is difficulty with acquiring new memories so that it is difficultto recall recent events. As Aβ progressed to worsened, one observesconfusion, irritability, aggression, language breakdown, mood swings,long-term memory loss, and the withdrawal of the person from societywhile their senses decline.

The cause of AD is yet to be determined, however there are varioushypothesis explaining the cause of this disease. One of the hypothesesproposed is Amyloid hypothesis, which is used in present invention.Amyloid hypothesis discloses the pathogenesis of AD to beessentially-related to beta amyloid peptide (BAP) (MW 4514 Da). Amyloidbeta is a peptide composed of 36-43 amino acids, which is processed froma transmembrane cellular glycoprotein amyloid precursor protein (APP).An autosomal dominant mutation in the APP results in a hereditary formof early-onset Alzheimer's disease, with increase in amyloid betapeptide. The amyloid beta peptide can form dimers which are quite toxicto neurons.

One of the experimental evidences supporting Amyloid hypothesis is fromthe genetic locus of the amyloid beta precursor protein (APP) onchromosome 21, patient with Down's syndrome (Trisomy 21) will have anextra gene copy for the APP, it is observed that these patientsuniversally succumb to AD by the age of 40 years. In addition,inheritable AD is also speculated to be involved with APOE4. Thisallelotype results in an excessive accumulation of beta amyloid in thebrain, preceding the very early appearance of AD at only 40 years ofage. Another research on transgenic mice expressing a mutation of thehuman APP gene shows that these mice develop fibrillar deposits ofamyloid peptide in the brain and develop Alzheimer's-like neuropathologywith specific spatial learning deficits.

As illustrated in FIG. 1, the APP protein located transmembranal withinplasma membrane and spanned from cytoplasm into the surroundingextracellular space. Post-translational processing of APP involvesseveral secretase enzymes to cleave APP into smaller polypeptides. It isnormal for a healthy person to have some beta amyloid peptide in his/hercerebrospinal fluid (CSF) and bloodstream as these peptides may diffuseout or be actively transported out from the brain; however people withAD have a pathologic accumulation of the beta amyloid peptide withintheir brains and are unable to excrete the peptide from the brain.

FIG. 2 shows the pathologic appearance of beta amyloid peptide in thebrain of persons with AD. As AD progresses, the beta amyloid peptideaggregates into fibrils and becomes deposited as insoluble precipitatein senile neuritic plaques, which may be stained histochemically and bedetected using magnetic resonance imaging (MRI).

Amyloid beta (Aβ) found to be the primary constituent of fibrils in theextraneuronal senile neuritic plaques of Alzheimer's patients' brains.It is believed that the mature fibrils of Aβ peptide are not neurotoxicper se, but may serve as a reservoir of small, diffusible solubleoligomers of Aβ. It has been shown that cortical levels of soluble Aβcorrelate with both the extent of synaptic loss and severity of theclinical symptoms.

Aβ₄₀ and Aβ₄₂ are two common isoforms of the peptide where Aβ₄₂ is morefibrillogenic, aggregates more quickly, and higher toxicity especiallyin the dimer state. Aβ has little toxicity while in the monomeric state.Therefore, inhibiting formation of soluble Aβ dimers will nullify theneurotoxicity and be effective in preventing and/or treating Alzheimer'sdisease.

Previous research has discovered that the addition of soluble Aβ dimersto rodent hippocampal slices will potently inhibit long-termpotentiation (LTP), enhance long-term depression (LTD), and reduce theneuronal dendritic spine density in the normal rodent hippocampus. Thesesoluble Aβ dimers are also known to interfere with memory of learningbehavior in rodents' brains.

Shankar's lab showed that the level of soluble Aβ-dimers is correlatedto the clinical Alzheimer's disease state and the degree of progressionof the dementia. It is known that the hippocampus is important in memoryformation and consolidation, the pathologic appearance of amyloidplaques correlates well with the progression of Alzheimer's disease andthe concomitant memory and cognitive losses. Clearance of these solubleAβ dimers from the brain will ameliorate the cognitive deficits.Researchers have theorized that dementia is resulted from clearance ofsoluble Aβ dimers in the brain.

It has also demonstrated that soluble Aβ dimers interfere withglutamatergic synaptic transmission, where mGluRs are necessary for LTDinduction. The data shows interference of soluble Aβ dimers with theNMDARs (N-methyl-D-aspartate receptors) is required for neuronaldendritic spine losses.

Multiple studies have shown that the soluble assemblies of oligomeric Aβare the neurotoxic species which cause the cognitive losses ofAlzheimer's disease. These oligomeric Aβ (oAβ) have also been calledAβ-derived diffusible ligands (ADDLs). Recent research has shown thatthe oAβ/ADDLs play a key role in cognitive decline.

Direct injection of oAβ/ADDLs into the hippocampi of live rats hascaused cognitive and memory losses characteristic of Alzheimer'sdisease. Gandy has shown that the brain levels of oAβ/ADDLs correlatewell with impaired acquisition of the Morris Water Maze (MWM) task byAPP^(E693Q) mice.

Selkoe's group has shown that Aβ dimers at subnanomolar concentrationsdirectly induce Tau hyperphosphorylation, disruption of the microtubulecytoskeleton and neuritic degeneration in the rat hippocampus. Othershave demonstrated that Aβ oligomers induce key characteristics of the ADphenotype including dendritic spine loss, altered hippocampal synapticplasticity, and impaired memory for learned behaviors.

The hypothesis that small soluble oligomers of Aβ underlie the keyphenotypic characteristics of Alzheimer's disease is supported byexperimental data showing that these small soluble oligomers may: (1)cause synaptic loss and decreased dendritic spine density; (2) causehyperphosphorylation of tau proteins with resulting intraneuronalneurofibrillary tangles and collapse of the neuritic cytoskeleton; (3)memory impairment and cognitive losses in the absence of amyloidplaques.

There are various ways AD diagnosis can be done; some of the examplesare behavioral and cognitive testing, and an MRI of the brain. Thepresence of senile plaques in brain MRI is the indicator used indiagnostic of AD. These senile plaques are composed of fibrils ofpolymerized and precipitated beta amyloid. The aggregated oligomers andfibrils formed are neurotoxic and can caused neuronal cell death, thusit is speculated to be key events in the pathogenesis and progression ofAD.

Most of the researches for Alzheimer's disease focus on preventingaccumulation of beta amyloid peptide in the brain, which is believed tobe a central event in the progression and pathogenesis of Alzheimer'sdisease. Some therapeutic strategies have been proposed, thesestrategies include but not limited to: (1) decreasing proteolyticproduction of Aβ through β-secretase inhibitors which block the firstcleavage of APP in extracellular; (2) γ-secretase inhibitors which blockthe second cleavage of APP in the cell membrane; (3) increasing theclearance of soluble Aβ dimers through immunotherapy to induce antibodyformation to amyloid beta; and (4) anti-aggregation drugs to inhibit theaggregation and polymerization of amyloid beta peptide.

Various natural plant-derived extracts or phytochemicals are beingtested to look for natural agents to prevent polymerization of Aβ.Natural agents such as apple procyanidins, grape-derived polyphenols,Resveratrol and polyphenol curcumin (diferuloylmethane) are found to beinhibiting Aβ aggregation. Other polyphenol compounds which inhibitβ-amyloid aggregation include: tannic acid, nordihydroguaiaretic acid,curcumin, rosmarinic acid, kaempferol, ferulic acid, (+)-catechin,(−)-epicatechin, and tetracycline. Blueberry-enriched polyphenols havebeen shown to prevent synaptic failure and adenosine triphosphateimbalance.

Flavonoids are also shown to have anti-aggregatory properties, exampleslike myricetin, chrysin, fisetin (3,3′,4′,7-tetrahydroxyflavone),3′,4′,7-trihydroxyflavone, 3,3′,4′-trihydroxyflavone,3,3′,7-trihydroxyflavone, 5-deoxykaempferol and synthetic derivatives.The glycosaminoglycan homotaurine (3-aminopropanesulfonic acid) also hasanti-aggregation properties, however this compound has failed Phase IIIclinical trials in US and therefore it is no longer in use clinically.

Oil palm (Elaies guineensis) has demonstrated various biological effectsin treating diseases and improving health condition using palm oil andother palm-based materials such as palm oil vegetation liquor and oilpalm phenolics.

Oil palm phenolics (OPP) is a complex aqueous derived from plant whichmainly comprises polyphenol compounds, shikimic acid, oligosaccharides,and lipid. In addition, gallic acid, protocatechuic acid,p-hydroxybenzoic acid, vanillic acid, caffeic acid, syringic acid,p-coumaric acid and ferulic acid are also presence in OPP throughhigh-performance liquid chromatography (HPLC), liquidchromatography-tandem mass spectrometry (LS/MS/MS) analyses. Five of themajor polyphenol compounds are protocatechuic acid, p-hydroxybenzoicacid, 3-caffeoylshikimic acid, 4-caffeoylshikimic acid, and5-caffeoylshikimic acid. These polyphenol compounds are known to havesignificant antioxidant properties as assayed by the DPPH(2,2-diphenyl-1-picrylhydrazyl) assay as well as the GAE (Gallic AcidEquivalent) assays.

Our prior research has demonstrated the neuroprotective effects of OPPin specific strains of mice, and therefore it is an object of presentinvention to disclose the efficiency of OPP in impeding or inhibitingthe aggregation of Aβ into the neurotoxic soluble Aβ dimers or oligomersin vitro.

Present invention discloses OPP containing some active compounds thatinhibit the aggregation of amyloid beta peptide and thus reduce thepolymerization of the peptide. This inhibition may cause the betaamyloid peptide to remain in the soluble monomeric state and facilitatethe clearance of the peptide from the brain via the normal physiologicmechanisms. Therefore, it is an object of present invention to provide acompound in preventing or slowing the progression of Alzheimer'sdisease.

Further objects and advantages of the present invention may becomeapparent upon referring to the preferred embodiments of the presentinvention as shown in the accompanying drawings and as described in thefollowing description.

SUMMARY OF THE INVENTION

Present invention discloses a composition comprising oil palm phenolics(OPP), wherein said composition is useful in preventing or treatment ofAlzheimer's disease and diseases related thereto. The composition isuseful in impeding aggregation of β-amyloid into neurotoxic Aβ-dimer.The composition is also useful in impeding polymerization of β-amyloid,impeding folding of β-pleated sheet secondary structure in β-amyloidpeptide and inhibiting kinetic accumulation of the β-pleated sheetamyloid peptide.

Present invention can be used in preparation of a medicament in atherapeutic effective amount for prevention or treatment of Alzheimer'sdisease and diseases related thereto in an individual in need thereof,which may be administered orally, conventional treatment forms,pharmaceutical formulations, nutritional supplement or as nutraceuticalsupplement.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, appended claims, and accompanying drawings, in which:

FIG. 1 shows the processing of beta amyloid peptide in the brain fromamyloid precursor protein (APP);

FIG. 2 shows the senile neuritic plaques of beta amyloid;

FIG. 3 shows the effect on molecular weights of beta amyloid peptideover time after treatment with oil palm phenolics (OPP);

FIG. 4a shows the effect on beta amyloid peptide aggregation aftertreatment without OPP;

FIG. 4b shows the effect on beta amyloid peptide aggregation aftertreatment with OPP;

FIG. 5 shows the fluorescence spectrogram of OPP at wavelength of 535nm;

FIG. 6 shows the Congo red dye binding curve at various concentrationsof OPP;

FIGS. 7a and 7b show the amide-I two-dimensional correlation spectra ofthe beta-amyloid samples incubated at 37° C. without OPP for 1 hour and10 hours respectively; and

FIG. 7c shows the amide-I two-dimensional correlation spectra of thebeta-amyloid samples incubated at 37° C. with OPP for 10 hours.

DETAILED DESCRIPTION OF THE INVENTION

The embodiments herein and the various features and advantageous detailsthereof are explained more fully with reference to the non-limitingembodiments that are illustrated in the accompanying drawings anddetailed in the following description. Descriptions of well-knowncomponents and processing techniques are omitted so as to notunnecessarily obscure the embodiments herein. The examples used hereinare intended merely to facilitate an understanding of ways in which theembodiments herein may be practiced and to further enable those of skillin the art to practice the embodiments herein. Accordingly, the examplesshould not be construed as limiting the scope of the embodiments herein.

Present invention provides oil palm phenolics (OPP) which constitutes awater soluble extract from the palm fruit (Elaies guineensis) and areeffective in treating Alzheimer's disease (AD) through inhibiting ofbeta amyloid peptides aggregation. The efficiency of this compound isvalidated by Mass Spectroscopy, Congo Red Binding Assay, and 2D-IRspectroscopy. These results demonstrate the anti-Alzheimer activity ofOPP providing initial evidence towards its potential therapeutic uses.

Best Mode For Carrying Out The Invention

The method of preparation and use of the present invention is furtherillustrated by the following experimental examples. It should beunderstood that these experimental examples, while indicating preferredembodiments of the invention, are given by way better elucidation only.A person skilled in the art can ascertain the essential characteristicsand embodiments of this invention, therefore various changes may beprovided to adapt to various usages and conditions.

Materials and Methods Preparation of Oil Palm Phenolics:

OPP is a vegetation liquor which is prepared from the oil-bearing palmfruit (Elaeis guineensis), by a proprietary method established by theMalaysian Palm Oil Board (MPOB) as described in the U.S. Pat. No.7,387,802. Briefly, oil palm fruits are crushed and squeezedmechanically, followed by removal of undissolved solids, oleaginousparts, colloids and higher molecular weight molecules from the remainingvegetation liquor which is an aqueous fraction containingphytochemicals. Oil palm phenolics comprise phytochemicals include butnot limited to polyphenols, flavonoids, phenolic acids, hydroxyl acids,oligosaccharides, lipids (fatty acids and oils), shikimic acid and metalions.

β-amyloid Peptide Sample Preparation

Predetermined amount of lyophilized β-amyloid powder was redissolved in50 ml of DMSO (dimethylsulfoxide) and 950 ml of PBS (phosphate-bufferedsaline, 10 mM, pH 7.4). Predetermined amount of lyophilized Ab42 powderwas pretreated and resolubilized with 69.5 μl of a mixture comprising ofCH₃CN/300 mM Na₂CO₃/250 mM NaOH (48.3:48.3:3.4, v/v/v). The resultingalkaline Ab42 solution (500 μM) was diluted 1:10 with phosphate buffer(10 mM, pH 7.7) containing NaCl (11 mM) to obtain a 50-μM Ab42 solutionat pH 8.0 and NaCl concentration of 10 mM.

The final assay conditions were as follows: 50 μM Ab42 in phosphatebuffer (8.7 mM) containing 10 mM NaCl, 14.5 mM Na₂CO₃, 0.85 mM NaOH, and8.2% ACN (acetonitrile, pH 8.0). The Ab42 solutions were incubated at30° C. without any stirring. Analyses were then performed at selectedtimes.

Aβ42 Aggregation Inhibition Studies (Congo Red Binding Assay)

This inhibition study was initiated by incubating Ab42 samples at 30° C.in assay variables of with and without OPP. The Ab42 samples areincubated without any stirring. OPP used in this study was solubilizedin water at a stock concentration of around 9%. In time courseexperiments, OPP stock solution was diluted in assay buffer to a finalconcentration of approximately 1.78% under assay conditions([OPP]/[Ab42]=1:5). Initially, aliquots of stock solution were added tothe Ab42 samples before incubation at 40° C. The inhibitory values wereread and recorded. IC₅₀ (half maximal inhibitory concentration) of thisstudy was calculated by dividing the full range of inhibitory values inhalf.

Thioflavin-T Fluorescence Assay

Thioflavin-T (ThT)-based fluorometric assays were performed with aspectrofluorometer using 96-well microtiter plates. The solutioncontaining Ab42 (50 μM) or Ab42 in the presence of ThT (1.5 μM) up to afinal volume of 2.0 ml is subjected to time scan in spectroflurometerafter incubation. The exciting emission was at a wavelength of 450 nmand the fluorescence emission signal was monitored at 535 nm (kex=446nm). The fluorescence intensity values at the plateau (300 s) wereaveraged after subtracting the background fluorescence from 1.5 μM ThTand tested compound.

Although the ThT is a well-known and well-used assay for beta amyloidpeptide aggregation, it is not possible to be used with OPP due to itsstrong fluorescent signal that overlaps with fluorescent signal fromThT. Thus ThT experiments were performed to examine the kinetics of betaamyloid peptide aggregation in the absence of OPP.

Congo Red Binding Assay

The stock solution of Ab42 (50 μM) was added into predetermined amountof OPP or phosphate buffer before diluted to a final concentration of 10μM. The mixture is incubated and added with 6 μL of Congo Red dye. TheCongo Red binding assays were performed with microplate reader and96-well microtiter plates. The absorption readings were monitored atboth wavelength of 492 nm and 540 nm, this is because the spectral shiftoccurs when Congo Red is bound to aggregated beta amyloid peptide. Thedata is then used to generate a bound Congo Red curve as described lateron.

Mass Spectroscopy by MALDI-TOF

Mass spectroscopy is useful in detecting and quantitating the molecularweights of polypeptides and their aggregates. The location and size ofthe peaks observed correlate well with the molecular weight and thequantity of substance present.

The analysis was performed by spotting on the target plate with 1.0 μlof the sample mixed with an equal volume of the matrix solution, 10mg/ml sinapinic acid, in CH₃CN/H₂O (50:50, v/v) containing 0.1% (v/v)trifluoroacetic acid. 10 μl of the sample was C4 ziptipped, eluted in 1μl of 70% ACN (acetonitrile), mixed with 1 μl of matrix, spotted, andallowed to air dry.

Ab42 MALDI-TOF (matrix-assisted laser desorption ionisation-time offlight) mass spectrometry analyses were performed using a MALDI-TOF massspectrometer equipped with a pulsed nitrogen laser operating at 337 nm.Small oligomer positive ion spectra were acquired in linear mode over anm/z range from 2000 to 50,000 using a 20-kV accelerating voltage and a150-ns delay extraction time. The spectrum for each spot was obtained byaveraging the results of 200 laser shots.

2-D FTIR Assay Equilibrium Measurements

Analogous to nuclear magnetic resonance (NMR) spectroscopy,two-dimensional infrared spectroscopy (2DIR) reveals structuralinformation onto two frequency axes and correlating the frequency ofinitial vibrational excitation (ω₁) with a final detection frequency(ω₃). The frequencies of the diagonal peaks correspond to thevibrational transitions within the sample, and cross peaks are onlyobserved when two vibrational modes are coupled (i.e. if the modesreside within the same structure or if there is energy transfer betweentwo vibrations). In a 2DIR spectrum, each positive diagonal peaks isaccompanied by a negative appearing below the diagonal, these negativepeaks are from vibrational transitions involving two-quanta states andcontain information related to the anharmonicities of the individualmodes.

Amyloid-beta samples were prepared in D2O at a concentration of 10 mg/mland buffered to a final pH of 7.4 in a 10 mM deuterated phosphate andheld between CaF2 windows in a 50 μm path-length cell. Spectra werecollected in the perpendicular (ZZYY) polarization geometry to enhancethe intensity of the cross peak.

Description of Spectra

In the amide-I region, beta-sheets are characterized by the presence oftwo peaks centered near 1620 and 1680 cm⁻¹, whose individual amideoscillators vibrate in-phase perpendicular (ν_(⊥)) or parallel (ν_(∥))to the β-strands, respectively. The splitting between these modes andthe frequency of ν_(⊥) in particular are related to the size of thefolded β-sheet. In a primarily β-sheet protein and the correspondingcross peaks give a characteristic Z-shape to the spectrum.

Present invention focuses on the cross peak centered at [w1, w3]=[1620,1680] cm⁻¹ whose amplitude which indicates the total amount of β-sheetpresent in the sample.

As shown in the experimental spectrum on FIGS. 7, the ν_(∥) peak ismasked by a strong diagonal peak at 1672 cm⁻¹, arising from the carbonylstretch in tetrafluoroacetic acid (TFA), a compound used to cleave thepeptide during the solid-state synthesis. TFA is particularly difficultto remove since lyophillization causes beta-amyloid aggregation. SinceTFA does not have any other transitions in the CO stretching region, theoff-diagonal features observed in the spectrum are only possible tocause by β-sheet structure. This describes the ability of 2DIRspectroscopy in extracting structural information even when the IRtransitions of interest are masked by other species present in solution.

The three spectra shown in FIGS. 7a, b and c are corresponded to theamyloid-beta sample incubated for a period of 1 hour and 10 hours at 37°C. in the absence of OPP, as well as incubated with OPP for 10 hours at37° C. respectively. The spectra show that the cross peak at [w1,w3]=[1620, 1680] cm⁻¹ (indicated by arrow) increases in amplitude overthis period. The increase in amplitude relates to an overall increase inβ-sheet content of the sample as well as the increase in growth ofβ-fibrils in within the sample.

Previous studies have shown that increasing incubation duration will notaffect amplitude of the cross peak. FIG. 7c shows sample incubated withOPP resulting in a very small cross peak even after incubation for 10hours. In FIGS. 7a and 7b , the diagonal peaks associated with β-sheetbecome significantly broader and signal near the 1650 cm⁻¹ regionincreases. Part of the spectrum is associated with helical andrandom-coil conformations, the absence of signal in FIG. 7c suggestingthat OPP disrupts the secondary structure of amyloids, and thuspreventing the formation of β-fibrils.

The 2DIR spectrum correlates the frequency of initial vibrationalexcitation (ω_(l)) with a final detection frequency (ω₃). Thefrequencies of diagonal peaks can be assigned to chemically distinctvibrational modes. The presence and splitting of cross-peakscharacterizes the anharmonic coupling of the vibrations and helpsdecompose congested spectra. Present invention concentrates on thediagonal and cross-peaks between two vibrational bands of β-sheets(ν_(⊥) and ν_(∥)), whose individual amide oscillators vibrate in-phaseperpendicular or parallel to the β-strands, respectively. The splittingbetween these modes and the frequency of β_(⊥) in particular areindicators of the size of the folded β-sheet and provide an importantsignature in transient experiments. Absorptive spectra were acquiredwith parallel (ZZZZ) and perpendicular (ZZYY) probing polarizations. Forβ-amyloid, the ν_(⊥) and ν_(∥) β-sheet modes are observed on 1620 cm⁻¹and 1680 cm⁻¹ regions of the amide I spectrum.

At 63° C., inhomogeneous broadening causes the two transitions to be notclearly resolved but appear as a broad diagonal peak both for thefundamental transition (ν=0→1, positive) and for the overtone transition(ν=1→2, negative). The overtone transition lies below the fundamentalalong the ω₃ axis because of the anharmonicity of the vibrationalpotential. The ν_(⊥) and ν_(∥) modes have nearly orthogonal transitionmoments where the cross-peaks are small in the parallel polarizationgeometry but are enhanced and form a cross-peak ridge in the upper leftcorner in the perpendicular polarization geometry. Loss of negativeintensity (a positive change) in the lower right corner also indicatesthe presence of a positive cross-peak in this region. The overallZ-shape of the perpendicular spectrum, which arises from interferenceeffects between ν_(⊥) and ν_(∥) diagonal and cross-peaks, is acharacteristic signature of the β-sheet structure.

Results

The mass spectrograms show that the beta amyloid peptide tend tospontaneously form aggregates consisting of dimers, trimers, tetramers,pentamers and higher order aggregates. In reference to FIGS. 3, 4 a and4 b, the presence of OPP impedes the aggregation process of beta-amyloidpeptide, and mostly the monomer beta amyloid peptide is observed withoutthe higher order aggregates.

Tabulation of Peak Aggregates Observed by Mass Spectroscopy

The molecular weights of the beta amyloid peptide and its aggregateswith or without the presence of OPP are shown in FIG. 3. There are 3species of peptides found in sample absence of OPP (0 mcg/ml) namely,monomer, dimer and trimer. At concentration of 0.9 mcg/ml OPP, only themonomer and dimer peptides are found. From the concentration of 90mcg/ml up to 90 mg/ml, only monomer of beta amyloid peptide is found.

FIGS. 4a and 4b show the raw data mass spectrograms of beta amyloidpeptide aggregation assay generated from the MALDI-TOF apparatus. FIG.4(a) shows beta amyloid peptides are aggregated up to septamers withmolecular weight of 31.5 kDa (7 peptides) in the absence of OPP, whereasin FIG. 4(b) only the monomer (4.5 kDa) of beta amyloid is observed inthe presence of OPP.

Inhibition of Beta Amyloid Peptide Aggregation by OPP

Beta amyloid peptide is incubated in PBS at 37° C. and maintained at pH7.4. FIG. 4(a) shows the aggregates of several molecular size forms,i.e. dimers, trimers, pentamers, hexamers and septamers, whereas in FIG.4(b) addition of OPP at a concentration of 0.9 mg/ml into the previouscondition inhibits the peptides from aggregating and remains in themonomeric form. The mass spectroscopy data clearly shows that thepresence of OPP will inhibit the aggregation and polymerization of betaamyloid peptide.

Thioflavin-T Dye Assay of Aggregation Dynamics and the Auto-FluorescenceOf Oil Palm Phenolics

Thioflavin-T assay is performed to observe the normal aggregationkinetics of the beta-amyloid peptide in the absence of OPP.

FIG. 5 shows a spectrogram of titrated concentrations of OPP fluorescingat a wavelength of 535 nm as similar to many antioxidant polyphenols.

The fingerprinting analysis of OPP (Sambandan et al.) reveals 5“signature” polyphenols present in OPP. These include p-hydroxybenzoicacid, protocatechuic acid, and 3 structural isomers of caffeoylshikimicacid. Based upon their spectroscopic properties, these 5 polyphenolcomponents of OPP most probably account for the strong autofluorescenceobserved at a wavelength of 535 nm.

Due to the strong autofluorescence of the OPP, it was not feasible toconduct aggregation or kinetic experiments on the β-amyloid peptide with“noise” of OPP fluorescence 3-4 times of higher magnitude than thefluorescent signal generated by the Thioflavin-T bound to aggregates.

Congo Red Binding Assay

The Congo Red assay is based on the spectral shift occurs in theabsorption of Congo Red bounded to beta amyloid peptide aggregates orfibrils at 2 different reference wavelengths.

In reference to FIG. 6, the dilution of OPP is directly proportional tothe amount of Congo Red bound to the peptide aggregates and oligomers.An inverse linear relationship between the amount of OPP solids and thequantity of beta amyloid aggregates can be observed. The degree of CongoRed binding relates to beta amyloid peptide aggregation, also show thatOPP inhibits the aggregation and polymerization of beta amyloid peptide.

IC₅₀ provides a good indication for the efficacy of inhibiting betaamyloid peptide aggregation by OPP. Based on FIG. 6 OPP inhibition ofCongo Red binding data, the IC₅₀ is 1.29 mg/ml of total solids by dryweight, at which point 23.22% of Congo Red is bound to aggregates.

Congo Red dye binds directly to the beta amyloid peptide and resultingin spectral shift when Congo Red bounded to aggregated peptide asopposed to monomeric peptide. The quantitation of aggregated betaamyloid peptide is given by the formula: Cb=A₅₄₁/47,800−A₄₀₃/38,100where Cb represents the amount of Congo Red dye bound to aggregatedpeptide, A₅₄₁ and A₄₀₃ are the optical absorptions measured at thewavelength of 541 nm and 403 nm respectively, 478000 and 38100 are theextinction coefficients at 541 nm and 403 nm respectively.

2DIR Results

FIGS. 7a, 7b and 7c show the Amide-I two-dimensional correlation spectraof the beta-amyloid samples incubated at 37° C. with and without OPP.The diagonal peak at 1672 cm⁻¹ is due to tetrafluoroacetic acid (TFA)present in the sample. The cross-peak near [w1, w3]=[1620, 1680] cm⁻¹ isdue the beta-fibrils. FIG. 7c shows broader peaks and a weaker crosspeak which indicates lower beta-fibril content.

From the results as disclosed in the description, OPP is shown tosubstantially impeding the aggregation of Aβ peptide into dimers by MassSpectroscopy, 2DIR spectroscopy and Congo Red dye binding assay.However, the active phytochemical agents in OPP that are useful ininhibiting formation of neurotoxic Aβ-dimers in present invention shouldnot be limited to one type.

From the result obtained from Congo Red binding assay, the IC₅₀(half-maximal inhibition) for OPP against beta amyloid peptideaggregation is approximately 1.29 mg/ml. The usual concentration of OPPis 9.0% solids, thus the concentration of solids is 90 mg/ml. The IC₅₀of 1.29 mg/ml is therefore a dilution of 1.43% of OPP (or a 1:70dilution of the OPP).

In addition, Mass Spectroscopy has provided very useful data showing theaggregation of the beta amyloid peptide monomer by revealing themolecular weights of the peptide species present. FIG. 4 shows theefficiency of OPP in impeding beta amyloid peptide aggregation whereamyloid-beta peptide aggregates and polymerize in the absence of OPP(FIG. 4a ) and amyloid-beta peptide remains monomeric in the presence ofOPP (FIG. 4b ).

The 2DIR spectroscopy also discloses the efficiency of OPP in inhibitingβ-amyloid peptide from misfolding into beta-pleated sheets on secondarystructure. In the absence of OPP, the kinetic accumulation of thebeta-pleated sheet amyloid peptide is shown in 2DIR spectroscopy,whereas in the presence of OPP there is no formation of beta-pleatedsheet structures.

Efficacy in maintaining beta amyloid peptide in soluble form andpreventing peptide from aggregating into insoluble polymers or fibrilsmay be important feature for a drug in prevention or treatment ofAlzheimer's disease. The peptides maintained in soluble form would allowexcretion with body's normal clearance mechanisms before it couldfibrillize into the senile neuritic plaques, and therefore preventingoccurrence of Alzheimer's disease.

It is possible that OPP binds to beta amyloid peptide, and prevents thehydrogen-bonding and hydrophobic interactions from polymerizing peptidein forming aggregated fibrils. 2DIR spectroscopic are conducted toelucidate the secondary and tertiary structure of this interactionbetween OPP and beta amyloid peptide.

The study of kinetics of aggregation for the 42-amino acid β-amyloidpeptide shows that (1) OPP impede β-amyloid monomers from aggregatingand polymerizing into larger aggregates as shown by the decrease inmolecular weights measured on Mass Spectroscopy; (2) OPP inhibit bindingof Congo Red dye to β-amyloid peptide as shown by the decrease in CongoRed dye binding to aggregated β-amyloid peptide; (3) OPP impede foldingof β-pleated sheet secondary structures, as revealed by 2DIRSpectroscopy.

It is well established that the soluble β-amyloid dimers and oligomerscause extensive pathologic changes in neurons, decrease dendritis spinedensity, and cause depression of long-term potentiation in neurons andenhancement of long-term depression. Therefore, the inhibitory,anti-aggregatory, and antifibrillogenic properties of OPP may lead tothe development of a potential drug for the prevention or treatment ofAlzheimer's disease.

The composition as disclosed in present invention may be provided ascompounds with pharmaceutically acceptable carriers. Present inventionfurther discloses the use of therapeutically effective amount of acomposition in the preparation of a medicament for preventing ortreatment of Alzheimer's disease or disease related thereto in anindividual by administering to an individual in need thereof. Thecomposition may be administered orally, conventional treatment forms,pharmaceutical formulations, nutritional supplement or as nutraceuticalsupplement.

It is understood by a person skilled in the art that the methods forexperiments and studies are described as exemplifications herein andthus the results are not intended, however, to limit or restrict thescope of the invention in any way and should not be construed asproviding conditions, parameters, agents, chemicals or startingmaterials which must be utilized exclusively in order to practice thepresent invention. It is therefore understood that the invention may bepracticed, within the scope of the appended claims, with equivalentmethods for the experiments than as specifically described and stated inclaims.

1. A composition comprising vegetation liquor, wherein said compositionis useful in preventing or treatment of Alzheimer's disease and diseasesrelated thereto.
 2. The composition as claimed in claim 1 wherein saidvegetation liquor is oil palm phenolics.
 3. The composition as claimedin claim 1 is useful in impeding aggregation of β-amyloid.
 4. Thecomposition as claimed in claim 2 wherein said β-amyloid aggregationwill lead to formation of neurotoxic Aβ-dimer.
 5. The composition asclaimed in claim 1 is useful in impeding polymerization of β-amyloid. 6.The composition as claimed in claim 1 is useful in impeding folding ofβ-pleated sheet secondary structure in β-amyloid peptide.
 7. Thecomposition as claimed in claim 1 is useful in inhibiting kineticaccumulation of the β-pleated sheet amyloid peptide.
 8. The compositionas claimed in claim 1 can be used in preparation of a medicament in atherapeutic effective amount.
 9. The composition as claimed in claim 1may be administered orally, conventional treatment forms, pharmaceuticalformulations, nutritional supplement or as nutraceutical supplement. 10.Use of therapeutically effective amount of a composition as claimed inclaim 1, in the preparation of a medicament for prevention or treatmentof Alzheimer's disease and diseases related thereto in an individual inneed thereof.